Table 2 Experimental methods for detecting m6A modification.
From: The role of N6-methyladenosine-modified non-coding RNAs in the pathological process of human cancer
Method | Name | Classification | Mechanism | Advantage | Deficiency | Reference (PMID) |
|---|---|---|---|---|---|---|
MeRIP | methylated RNA immunoprecipitation sequencing | Rely on m6A antibody | MeRIP enriched the m6A-modified fragment with an anti-m6A antibody incubated with the RNA fragment for high-throughput sequencing detection of m6A | MeRIP is an earlier method for detecting m6A and facilitates the research progress of m6A | MeRIP relies on the specificity of anti-m6A antibody with a rather low resolution (at least 100nt) | 22608085 22575960 |
PA-m6A-seq | photo-cross-linking-assisted m6A sequencing strategy | Rely on anti-m6A antibody | PA-m6A-seq metabolically incorporates 4SU into RNA and covalently cross-links 4SU with an aromatic amino acid residue adjacent to the anti-m6A antibody upon 365 nm UV irradiation | PA-m6A-seq increases the resolution of m6A up to about 23nt | PA-m6A-seq can only be used in cells due to the metabolism of 4SU | 25491922 |
miCLIP | m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation | rely on m6A antibody | The miCLIP cross-links RNA with anti-m6A antibody using 254 nm irradiation | Achieving the single nucleotide resolution for detecting m6A at transcriptome-wide level | The miCLIP identifies a limited number of m6A sites because of the low cross-linking efficiency | 26121403 34157120 |
m6A-LAIC-seq | m6A-level and isoform-characterization sequencing | rely on m6A antibody | Utilize sequencing method in complete full-length RNAs after RNA Binding Protein Immunoprecipitation Assay by anti-m6A antibody | Quantitate m6A at transcriptome level | Mainly used to distinguish methylated from non-methylated transcripts | 22575960 24713629 24981863 27376769 |
MAZTER-Seq/m6A-REF-seq | m6A-sensitive RNA-endoribonuclease-facilitated sequencing | m6A antibody independent | Endoribonuclease-based strategies to detect m6A | They can accurately detect m6A modifications at that single nucleotide level | MazF can only recognize specific m6A motifs (ACA), so their detection efficiency is low | 31257032 31281898 |
DART-Seq | Deamination adjacent to RNA modification targets sequencing | m6A antibody independent | The fusion APOBEC1-YTH protein induced single nucleotide mutation in the adjacent site of m6A (C to U), so that m6A modification could be detected | DART-seq is efficient in detection and capable of detecting m6A modifications accumulated over time in cells | The binding ability between the fusion APOBEC1-YTH protein and m6A modifications may affect the detection accuracy of the targets | 31548708 |
m6A-label-seq | A metabolic labeling method detects m6A | m6A antibody independent | The m6A-label-seq chemically labels intermediates during the biogenesis of m6A and allows the detection of m6A at a single base resolution level | The m6A-label-seq can recognize various m6A motifs at a single base resolution level | The m6A-label-seq only can be used in cellular system | 32341503 32341502 |
m6A-SEAL | FTO-assisted m6A selective chemical labeling method | m6A antibody independent | The m6A-SEAL that couples FTO’s enzymatic oxidation of m6A to the unstable intermediate hm6A with a DTT-mediated thiol-addition reaction to generate a more stable dm6A with a sulfhydryl group, which could detect m6A effectively | The m6A-SEAL has higher sensitivity and specificity | Single base resolution is not yet implemented | 32341503 32341502 |
